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Advances in monolithic silica columns for high-performance liquid chromatography. Gaurav Sharma , Anjali Tara , Vishnu Dutt Sharma. Journal of Analyti...
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Anal. Chem. 2006, 78, 7632-7642

Performance of Monolithic Silica Capillary Columns with Increased Phase Ratios and Small-Sized Domains Takeshi Hara, Hiroshi Kobayashi, Tohru Ikegami, Kazuki Nakanishi, and Nobuo Tanaka*

Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan, and Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of ∼1.3 µm and silica skeletons of ∼0.9 µm) showed a plate height of less than 5 µm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of ∼1, and ∼7 µm at u ) 8 mm/s. With a permeability similar to that of a column packed with 5-µm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-µm particles, and showed performance in the “forbidden region” for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15 00030 000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns. Monolithic silica columns have been reported to have some advantages over silica particle-packed columns, including higher permeability at a similar column efficiency and higher mechanical stability.1-3 Monolithic silica capillary columns can be used without the frits that are required for a particulate column to retain their packing. For monolithic silica columns prepared in a test tube * Corresponding author. E-mail: [email protected]. Phone: 81-75-724-7809. FAX: 81-75-724-7710. (1) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. Anal. Chem. 1996, 68, 3498-3501. (2) Tanaka, N.; Kobayashi, H.; Nakanishi, K.; Minakuchi, H.; Ishizuka, N. Anal. Chem. 2001, 73, 420A-429A. (3) Motokawa, M.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.;Nakanishi, K.; Jinnai, H.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr., A 2002, 961, 53-63.

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and clad with PEEK resin, plate heights similar to that of a column packed with 3.5-4-µm silica particles were typically obtained at a pressure drop similar to that for a column packed with 7-10-µm particles.4,5 Monolithic silica capillary columns with large-sized domains (the domain size is defined as the combined size of a through-pore and a skeleton) showed up to 30-fold higher permeability than a column packed with 5-µm particles to produce more than 100 000 theoretical plates at a low-pressure drop.6 The separation impedance shown by such monolithic silica columns was found to be much smaller than that for particle-packed columns by a factor of 5 or more.7 This can be attributed to the high porosity and large through-pore size with small-sized skeletons, leading to large through-pore size/skeleton size ratios that were made possible by the integrated network structures. The through-pore size/skeleton size ratios are in the range of 0.25-0.4 for a particle-packed column,8 whereas ratios of 1-4 were obtained with monolithic silica columns.6 The small-sized skeletons facilitated a stationary-phase mass transfer,4,9 although a greater contribution of the slow mobile-phase mass transfer was observed due to the larger sized through-pores.6 It has not been possible, however, to prepare monolithic silica columns with smaller sized domains that can show performance similar to advanced columns packed with silica particles of ∼2 µm or smaller. When compared with columns packed with small silica particles of 1.7-2-µm diameter, previous monolithic silica columns were not competitive in high-speed separations in a range generating ∼50 000 theoretical plates or less, with a limiting pressure drop of 40 MPa.10 It has been pointed out that there were problems in the homogeneity and uniformity of the skeletons and through-pores of monolithic silica columns prepared to have smallsized domains.11 Such monolithic columns showed inhomogeneous structures consisting of skeletons with the appearance of (4) Miyabe, K.; Cavazzini, A.; Gritti, F.; Kele, M.; Guiochon, G. Anal. Chem. 2003, 75, 6975-6986. (5) Leinweber, F. C.; Tallarek, U. J. Chromatogr., A 2003, 1006, 207-228. (6) Ishizuka, N.; Kobayashi, H.; Minakuchi, H.; Nakanishi, K.; Hirao, K.; Hosoya, K.; Ikegami, T.; Tanaka, N. J. Chromatogr., A 2002, 960, 85-96. (7) Tanaka, N.; Kobayashi, H.; Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Hosoya, K. Ikegami, T. J. Chromatogr., A 2002, 965, 35-49. (8) Unger, K. K. Porous Silica; Journal of Chromatography Library 16; Elsevier: Amsterdam, 1979; Chapter 5. (9) Kobayashi, H.; Tokuda, D.; Ichimaru, J.; Ikegami, T.; Hosoya, K.; Miyabe, K.; Tanaka, N. J. Chromatogr., A 2006, 1109, 2-9. (10) Desmet, G.; Clicq, D.; Gzil, P. Anal. Chem. 2005, 77, 4058-4070. 10.1021/ac060770e CCC: $33.50

© 2006 American Chemical Society Published on Web 10/12/2006

Figure 1. Scanning electron micrographs of monolithic silica capillary columns with increased phase ratios. Scale bars correspond to 20 µm. Circles show examples of agglomerated skeletons in (a).

Table 1. Composition of the Feed Mixtures for the Preparation of Monolithic Silica Columns column

TMOS (mL)

PEG (g)

urea (g)

CH3COOH (mL)

MS(100)-T1.0-Aa MS(100)-T1.4-Ab MS(100)-T1.6-Ab MS(100)-T1.8-Ab MS(100)-T1.0-Ba MS(100)-T1.4-BIb MS(100)-T1.4-BIIb MS(100)-T1.4-BIIIb

40 56 64 72 40 56 56 56

12.4 11.8 10.4 8.4 12.8 11.7 11.8 11.9

9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0

100 100 100 100 100 100 100 100

a

Prepared at 30 °C. b At 25 °C.

agglomerated particles as well as the presence of through-pores of various sizes, resulting in lower performance than expected.3 High porosity can increase performance over a region of large numbers of theoretical plates just like a column packed with large particles, but not for a high linear velocity region. Another problem found with previous monolithic silica columns was small phase ratios, resulting in small k values.12 Small phase ratios lead to small k values, which in turn lead to low resolutions in a strong mobile phase. Sometimes a problem was observed for large-volume injections or the injection of strong solvents.13 (11) Gzil, P.; Vervoort, N.; V. Baron, G.; Desmet, G. Anal. Chem. 2004, 76, 67076718.

Desmet and co-workers suggested that increased phase ratios and increased homogeneity of the skeletons and throughpores might increase the total performance of monolithic silica columns by severalfold.11 The sizes of the skeletons and throughpores of monolithic silica columns should be reduced, and the homogeneity increased, to attain the performance desired. Here we report the results of the preparation of monolithic silica capillary columns with increased phase ratios and small-sized domains and the chromatographic characterization of these columns. The evaluation proposed by Desmet and co-workers10 was carried out to show a comparison against particle-packed columns. EXPERIMENTAL SECTION Materials. Tetramethoxysilane (TMOS), octadecyldimethylchlorosilane (ODS-Cl, Shinnetsu Chemicals), poly(ethylene glycol) (PEG; Mn 10 000) (Aldrich), urea, acetic acid (Wako Pure Chemicals), and diethylamine (DEA) (Nacalai Tesque) were obtained from commercial sources. A column packed with 5-µm ODS-silica particles, Mightysil RP18, was provided by Kanto Chemical. Fused-silica capillaries of 100- and 200-µm i.d. and 375µm o.d. were purchased from Polymicro Technologies. Preparation of Monolithic Silica Columns. The preparation conditions of the monolithic silica columns were similar to those (12) Ishizuka, N.; Minakuchi, H.; Nakanishi, K.; Soga, N.; Nagayama, H.; Hosoya, K.; Tanaka, N. Anal. Chem. 2000, 72, 1275-1280. (13) Ikegami, T.; Dicks, E.; Kobayashi, H.; Morisaka, H.; Tokuda, D.; Cabrera, K.; Hosoya, K.; Tanaka, N. J. Sep. Sci. 2004, 27, 1292-1302.

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Figure 2. Scanning electron micrographs of monolithic silica capillary columns with increased phase ratios and smaller domain sizes. Scale bars correspond to 50 µm for (a)-(d) and to 20 µm for (e)-(h).

reported earlier.3 The typical conditions are as follows. A fusedsilica capillary tubing (2-3 m in length) was treated with a 1 N aqueous sodium hydroxide solution at 40 °C for 3 h, washed with water and acetone, and then dried. TMOS (40 mL) was added to a solution of PEG (12.4 g) and urea (9.0 g) in 0.01 M acetic acid (100 mL) and stirred at 0 °C for 30 min. The resultant homogeneous solution was charged into a fused-silica capillary tube and allowed to react at 25 or 30 °C. The resultant gel was subsequently 7634

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aged in the capillary overnight at the same temperature. Then, the temperature was raised, and the monolithic silica column was treated for 3 h at 120 °C to form mesopores with ammonia generated by the hydrolysis of urea, followed by water and methanol washes. After drying, a heat treatment was carried out at 330 °C for 25 h, resulting in the decomposition of the organic moieties in the capillary. Surface modification of the monolithic silica was carried out on-column by continuously feeding a solution

Table 2. Domain Size and Permeability Observed for Monolithic Silica Columns with Increased Phase Ratios and Different Domain Sizes

column

skeleton size (µm)

throughpore size (µm)

domain size (µm)

MS(100)-T1.0-A MS(100)-T1.4-A MS(100)-T1.6-A MS(100)-T1.8-A MS(100)-T1.0-B MS(100)-T1.4-BI MS(100)-T1.4-BII MS(100)-T1.4-BIII Mightysil RP18

1.5 1.0 1.3 1.2 1.2 1.3 1.0 0.9 (5.0)

2.1 1.6 1.5 1.3 1.8 1.8 1.6 1.3

3.6 2.6 2.8 2.5 3.0 3.1 2.6 2.2

permeability K (×10-14 m2)a 11.1 5.4 4.1 1.3 7.4 10.1 5.9 4.7 4.8

a Permeability was measured in methanol-water (80/20) at 30 °C for the A-series columns and MS(100)-T1.0-B of 100 µm i.d. × 25 cm (effective length 20 cm), MS(100)-T1.4-BI of 19.5 cm (effective length 14.5 cm), MS(100)-T1.4-BII and MS(100)-T1.4-BIII of 20 cm (effective length 15 cm), and a Mightysil RP18 column of 4.6 mm i.d. × 15 cm.

of octadecyldimethyl-N,N-diethylaminosilane (ODS-DEA prepared from ODS-Cl and DEA, 2 mL) in 8 mL of toluene driven by a nitrogen pressure of 5 bar at 60 °C for 3 h. As shown in Table 1, the PEG concentration was varied along with the TMOS concentration. The morphology of the monolithic silica was examined by a scanning electron microscope (SEM; S-510, Hitachi) using a fractured surface. The through-pore size and skeleton size were measured from the photographs by averaging the sizes of more than 50 through-pores or skeletons clearly observed. A series of columns designated as MS(100)-T1.0-A, MS(100)T1.4-A, MS(100)-T1.6-A, and MS(100)-T1.8-A were used for the pore size characterization by size exclusion chromatography (SEC) with and without surface modification. The abbreviation, MS, stands for monolithic silica followed by the capillary diameter in parentheses, and T for the starting material, TMOS, followed by numbers indicating the extent of the increase in TMOS concentration in the feed as compared to the previous preparations.3 As shown in Table 1, another B-series of columns, MS(100)T1.0-B, MS(100)-T1.4-BI, MS(100)-T1.4-BII, and MS(100)-T1.4BIII, were prepared with TMOS concentrations 1.0 or 1.4 times compared to the previous preparations to examine the effects of domain size on column performance at high speed. Instrument and Chromatographic Measurements. Two sets of HPLCs were used for the characterization of the monolithic silica capillary columns. One set consisted of a MP 681 pump (GL Sciences), C4-00R-0.01 10-nL injector (Valco), and CE1575 detector (Jasco) for SEC, and the other set was a LC-10AD VP (Shimadzu) with a split injection/flow mode using a Rheodyne 7725 (Rheodyne) and CE-2075 UV detector (Jasco) for the other chromatographic measurement. The chromatographic measurements using split injection/flow mode were performed as previously described.6 The chromatographic data were processed with EZChrom Elite software (GL Sciences). SEC was carried out in tetrahydrofuran (THF) in order to characterize the pore properties of the monolithic silica columns using polystyrene standards (Chemco). RESULTS AND DISCUSSION SEM Observation. Two series of feed compositions were employed to produce monolithic silica columns with various

Figure 3. Selective permeation of polystyrene standards in THF observed for columns with increased phase ratios. Squares, MS(100)T1.0-A; diamonds, MS(100)-T1.4-A; and triangles, MS(100)-T1.6-A. The solid symbols represent silica columns, and the open symbols represent ODS-modified monolithic silica columns.

domain sizes and phase ratios. For the A-series columns, the amount of TMOS in the starting mixture was varied together with the PEG concentration. The SEM photographs of the monolithic silica prepared in a 100-µm capillary tube are shown in Figure 1. The monolithic silica columns prepared with the increased TMOS concentration in the starting mixture, MS(100)-T1.4-A, MS(100)T1.6-A, and MS(100)-T1.8-A, were shown to have reduced porosity. Greater homogeneity in the skeleton structures of these columns (Figure 1b-d) than the product from the previous preparation method, MS(100)-T1.0-A (Figure 1a), was also noticeable. Although the MS(100)-T1.0-A column possessed agglomerated skeletons, those with increased phase ratios showed spongy structures. Another series of columns, MS(100)-T1.4-BI, MS(100)-T1.4BII, and MS(100)-T1.4-BIII, were prepared at a constant TMOS concentration and different PEG concentrations so that the product could have a larger or smaller domain size. The TMOS concentration was selected because of the higher success ratio (50-80% for MS(100)-T1.4-BI, MS(100)-T1.4-BII, and MS(100)-T1.4-BIII) of preparation than the other concentrations tested. Actually, MS(100)-T1.0-A and MS(100)-T1.0-B were prepared to replicate previous preparations. The preparation conditions for MS(100)T1.0-B were found to yield a smaller domain size than those for MS(100)-T1.0-A. As shown in Figures 1 and 2, monolithic silica columns with a smaller domain size can be prepared at a higher TMOS concentration. When compared to MS(100)-T1.0-A and MS(100)-T1.0-B, those columns prepared with a higher concentration of TMOS at a lower temperature possessed more evenly distributed skeletons, as shown in Figures 1b-d and 2f-h. MS(100)T1.4-BIII had the smallest domain size among the monolithic silica columns produced. Skeleton size, through-pore size, and the permeability, represented by a K value (K ) uηL/∆P, where u stands for the linear velocity of the mobile phase, η for solvent viscosity, L for column length, and ∆P for pressure drop), were measured for the monolithic silica columns and shown in Table 2. The permeability Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Table 3. Pore Volumes Measured by Size Exclusion Chromatography Using Polystyrene Standard Samplesa

column

total porosity (Vm)

throughpore (V0)

mesopore

MS(100)-T1.0-A(silica) MS(100)-T1.0-A(ODS) MS(100)-T1.4-A(silica) MS(100)-T1.4-A(ODS) MS(100)-T1.4-BII(ODS MS(100)-T1.6-A(silica) MS(100)-T1.6-A(ODS)

0.950 0.928 0.938 0.898 0.909 0.924 0.880

0.761 0.760 0.689 0.679 0.696 0.646 0.636

0.189 0.168 0.249 0.219 0.213 0.278 0.244

bonded phase (Vs)

phase ratio (Vs/Vm)

k(hexylbenzene)b

R(CH2)b

0.022

0.024

1.39

1.50

0.040

0.045

2.16 2.08

1.50 1.50

0.044

0.050

2.57

1.50

a Mobile phase, THF. Temperature, 30 °C. The same columns were used for the permeability measurement. b Measured in methanol-water (80/20) at 30 °C. The ratio of the retention factors was calculated as R(CH2) ) k(hexylbenzene)/k(amylbenzene).

Figure 4. Chromatograms obtained for uracil (the first peak that was used to obtain t0) and alkylbenzenes (C6H5(CH2)nH, n ) 0-6). Column: (a) MS(100)-T1.0-A, (b) MS(100)-T1.4-A, (c) MS(100)-T1.6-A, and (d) MS(100)-T1.8-A. Column size: 100 µm i.d. × 25 cm (effective length 20 cm). Mobile phase: methanol/water ) 80/20. Temperature: 30 °C. The pressure drop, linear velocity, and number of theoretical plates and the k value for hexylbenzene (the last peak) are indicated.

was measured in 80% methanol after ODS-DEA modification. The agglomerated skeletons (examples circled in Figure 1a) were excluded from the measurement of the pore size and skeleton size using SEM photographs. A decrease in the permeability was observed with an increase in the phase ratio for the A-series columns. With the increase in the PEG concentration of the feed, a smaller domain size and lower permeability were observed for MS(100)-T1.4-BI-MS(100)-T1.4-BIII. Figure 3 shows the relationship between elution volume in THF and the molecular weight of a polystyrene standard sample 7636

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obtained for the columns MS(100)-T1.0-A, MS(100)-T1.4-A, and MS(100)-T1.6-A with and without ODS modifications. The measurement for MS(100)-T1.8-A was omitted because of a highpressure drop and poor efficiency observed with this column. The total porosity was found to be ∼95% for MS(100)-T1.0-A-silica, which is similar to previous results,12 and ∼92% for MS(100)-T1.6A-silica. The difference presumably reflects the difference in the phase ratio. Selective permeation was observed for a wide range of polystyrene standard samples, as previously reported,14 indicating wide pore-size distributions associated with these monolithic

Figure 5. Chromatograms obtained for uracil (the first peak) and alkylbenzenes (C6H5(CH2)nH, n ) 0-6). Column: (a) MS(100)-T1.0-B, 25 cm (effective length 20 cm), (b) MS(100)-T1.4-BI, 19.5 cm (effective length 14.5 cm), (c) MS(100)-T1.4-BII, 20 cm (effective length 15 cm), and (d) MS(100)-T1.4-BIII, 20 cm (effective length 15 cm). Column diameter: 100 µm. Mobile phase: acetonitrile/water ) 80/20. Temperature: 30 °C. The pressure drop, linear velocity, and number of theoretical plates for hexylbenzene (the last peak) are indicated.

silica columns. A polystyrene standard sample with a molecular weight of 2 × 106 and benzene were used for determining the through-pore volume and total permeation volume, respectively. The mesopores of the monolithic silica capillary columns seemed to be distributed in a size range of up to 50 nm according to the comparison with the results reported for columns packed with particles of various pore sizes.15 The pore volumes measured are summarized in Table 3. Through-pores accounted for ∼65-76% of the column volume for silica columns without ODS modifications. With the increase in TMOS concentration in the feed, smaller through-pore volumes and greater mesopore volumes were observed. The difference in the pore volume caused by the ODS modification was taken as the volume of the stationary phase, or bonded alkylsilyl moieties. The phase ratio was calculated by dividing the volume of the bonded phase by the total pore volume obtained from the elution volume of benzene. An increase in the phase ratio was achieved with an increase in the TMOS concentration in the feed. The chromatograms in Figure 4 show the performance of the A-series monolithic silica columns after ODS modifications with a difference in the phase ratio for alkylbenzenes in 80% methanol. Symmetrical peaks were obtained for the monolithic columns. (14) Al-Bokari, M.; Cherrak, D.; Guiochon, G. J. Chromatogr., A 2002, 975, 275284. (15) Kirkland, J. J. J. Chromatogr. 1976, 125, 231-250.

Figure 6. Column back pressure observed for ODS-modified monolithic silica columns against the linear velocity of the mobile phase. Mobile phase: acetonitrile/water ) 80/20. The pressures were normalized to a column length of 15 cm. Columns: Mightysil RP18 (O), MS(100)-T1.0-B ([), MS(100)-T1.4-BI (2), MS(100)-T1.4-BII (0), and MS(100)-T1.4-BIII (X).

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Figure 8. Separation impedance against the linear velocity of the mobile phase calculated for hexylbenzene as the solute. Mobile phase: acetonitrile/water ) 80/20. Temperature: 30 °C. The symbols are the same as in Figure 6 for the columns.

Figure 7. van Deemter plots obtained for ODS-modified monolithic silica columns and a silica-C18 packed column with ethylbenzene (a) and hexylbenzene (b) as solutes. Mobile phase: acetonitrile/water ) 80/20. Temperature: 30 °C. The symbols are the same as in Figure 6 for the columns.

Figure 4 shows that MS(100)-T1.4-A and MS(100)-T1.6-A columns showed higher column efficiency in addition to a larger retention factor than MS(100)-T1.0-A, at the expense of an increased pressure drop, as expected for columns with smaller sized through-pores and lower porosity. However, the preparation conditions for the monolithic column with the highest phase ratio, MS(100)-T1.8-A, were considered to be unsatisfactory based on the low permeability and low column efficiency of the product. Although a ∼10% reduction in column efficiency is expected based on the lower linear velocity employed for this column, a greater decrease in column efficiency was observed. A fair agreement was observed between the phase ratios estimated from the SEC measurement in THF and the retention factors for alkylbenzenes in 80% methanol in reversed-phase mode. The retention factors for alkylbenzenes in reversed-phase mode can reflect the carbon content of the stationary phase, which is determined by the surface 7638 Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

area of the silica support and surface coverage.16 The surface coverage of the three stationary phases seems to be similar, as indicated by similar R(CH2) values.16 Figure 5 shows the effect of the domain size of a monolithic silica column on the performance of ODS-modified MS(100)-T1.4BI, MS(100)-T1.4-BII, and MS(100)-T1.4-BIII columns with increased phase ratios. Columns with a smaller domain size showed an increase in column efficiency, as expected. MS(100)-T1.4-BIII showed comparable permeability to a column packed with 5-µm particles (Table 2), while giving a plate height of 4.8 µm at u ) 2 mm/s for hexylbenzene with a retention factor of ∼1.1. Figure 6 shows the plots of the column pressure drop against the linear velocity of a mobile phase, 80% acetonitrile. A decrease in permeability was observed with a decrease in the domain size. Although the permeability of MS(100)-T1.4-BI was about twice as high as that of a column packed with 5-µm particles, the permeability of MS(100)-T1.4-BIII was comparable with the latter. The fact that greater permeability and higher column efficiency were observed simultaneously for MS(100)-T1.4-BI, which has a greater amount of skeletons than MS(100)-T1.0-B, suggests that MS(100)-T1.4-BI possessed greater structural homogeneity than the product of the previous preparations. MS(100)-T1.4-BII and MS(100)-T1.4-BIII showed a slight increase in column efficiency and pressure drop. Increased structural homogeneity for MS(100)T1.4-BI, MS(100)-T1.4-BII and MS(100)-T1.4-BIII as compared to MS(100)-T1.0-B can also be observed in Figure 2f-h. Panels a and b in Figure 7 show the plots of plate height (H) against linear velocity of a mobile phase for the elution of ethylbenzene and hexylbenzene, respectively. The smaller minimum plate height and the shift of the optimum linear velocity toward a higher value were observed with the decrease in domain size. The plots for MS(100)-T1.4-BIII showed a plate height of 4.1 µm for ethylbenzene and 4.7 µm for hexylbenzene at each optimum linear velocity. Such plate height values can be expected for a column packed with ∼2-2.5-µm silica particles. The domain size of the monolithic silica column was similar to the size of (16) Kimata, K.; Iwaguchi, K.; Onishi, S.; Jinno, K.; Eksteen, R.; Hosoya, K.; Araki, M.; N. Tanaka, J. Chromatogr. Sci. 1989, 27, 721-728.

Figure 9. Chromatograms obtained for uracil (the first peak) and alkylbenzenes (C6H5(CH2)nH, n ) 0-6) at increased flow rates. Column: MS(100)-T1.4-BII, 20 cm (effective length 15 cm). Column diameter: 100 µm. Mobile phase: acetonitrile/water ) 80/20. Temperature: 30 °C. The pressure drop, linear velocity, number of theoretical plates, and t0 are indicated.

particles that would have been expected to show a similar column efficiency.4,17 Although the column efficiency was somewhat lower than that of a column packed with 1.7-µm particles or smaller,18-20 the results showed that it is possible to prepare monolithic silica columns of increased column efficiencies by increasing the phase ratio or structural homogeneity, as predicted.11 The slightly greater increase in plate height at a higher linear velocity than that of a column packed with 1.5-1.7-µm particles can be attributed to the presence of relatively large through-pores that should increase the effect of slow mass transfer in the mobile phase. The advantage with the monolithic silica columns is that performance similar to that of a column packed with 2-2.5-µm particles can be obtained at a pressure drop similar to a column packed with 5-µm particles. Figures 6 and 7 suggest that the gain in column efficiency compared to a column packed with 5-µm particles was slightly greater than 2-fold at a similar pressure drop and at a similar linear velocity. Despite the high column efficiency at the (17) Minakuchi, H.; Nakanishi, K.; Soga, N.; Ishizuka, N.; Tanaka, N. J. Chromatogr., A 1997, 762, 135-146. (18) MacNair, J. E.; Lewis, K. C.; Jorgenson, J. W. Anal. Chem. 1999, 71, 849856. (19) Nova´kova´, L.; Matysova´, L.; Solich, P. Talanta 2006, 68, 908-918. (20) de Villiers, A.; Lauer, H.; Szucs, R.; Goodall, S.; Sandra. P. J. Chromatogr., A 2006, 1113, 84-91.

relatively low pressure drop of the B-series monolithic silica capillary columns, the results from Figures 5 and 7 suggest that the performance of MS(100)-T1.4-BII and MS(100)-T1.4-BIII columns was still not as high as expected based on their domain sizes compared to the MS(100)-T1.4-BI column. Figure 8 shows a plot of the separation impedance (an E value, E ) H2/K ) (t0/N)(∆P/N)(1/η)), or total performance of a column based on column efficiency and permeability, against the linear velocity with hexylbenzene as a solute. The smallest E values were obtained for the MS(100)-T1.4-BI column, which was ∼1/10 of that for the Mightysil column packed with 5-µm particles. The E values observed for MS(100)-T1.4-BII and MS(100)-T1.4BIII were larger than those for MS(100)-T1.4-BI by a factor of ∼2, but smaller than those for a particle-packed column by a factor of 5. The results suggest that the preparation conditions were not yet optimized for the monolithic silica with the smallest domain size. The increase in the E value associated with the decrease in domain size was still significant but actually much less than that observed for the MS(50)-D column in the previous study.3 The column efficiency of MS(100)-T1.4-BII at a high linear velocity of the mobile phase (Figure 9), showing H ) 7 µm at u ) 8 mm/s, was much better than those reported for previous monolithic silica columns. Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Figure 10. Log(t0/N) values against log(N) for the columns evaluated. The curves were obtained by assuming the following parameters: maximum pressure 20 MPa, η ) 0.00046 Pa s, φ ) 700, Dm ) 2.22 × 10-9 m2/s, and Knox equation h ) 0.65ν1/3+2/ν + 0.08ν. The particle diameters for the particle-packed columns were 1.4, 2, 3, and 5 µm. The dashed lines indicate the required t0 values in seconds. A previous monolithic silica column, MS(50)-D is indicated by (]). The other symbols are the same as in Figure 6 for the columns.

Figure 10 shows plots of the log(t0/N) values against log(N),21 a Poppe plot for the columns evaluated, where t0 is the column dead time and N is the number of theoretical plates. This plot provides a comparison of the performance of various types of columns in terms of the attainable N and t0 at a specified pressure drop. The plots in Figure 10 were made at a pressure limit of 20 MPa, because most HPLC separations have been carried out at a pressure of 20 MPa or lower. The comparisons at 40 MPa and other pressures are shown later. The curves for the particle-packed columns were calculated using A ) 0.65, B ) 2, and C ) 0.08 for the Knox equation,22 h ) Aν1/3 + B/ν + Cν (h stands for the reduced plate height, ν for reduced velocity, and A, B, and C for coefficients for the contribution of each term), which demonstrates a higher column efficiency than those employed by Poppe at A ) 1.0, B ) 1.5, and C ) 0.05.21 The equation used in this study fits the performance of Mightysil-RP18, which showed very high performance as a column packed with 5-µm particles (the Mightysil column gave an optimal E value below 2000). Figure 10 shows that the performance of a monolithic silica capillary column of the smallest domain size in the previous report3 (MS(50)-D) merges with the plots of the particle-packed columns at a t0 of ∼130 s, or ∼50 000 theoretical plates, while it may give a higher limiting N value. The present monolithic silica column with the smallest domain size, MS(100)-T1.4-BIII, was shown to give higher performance than a column packed with 3)µm particles for most of the range shown, and higher performance than a particulate column with a 2)µm particle size at around t0 ) 10 s or above and in the range of ∼15 000 theoretical plates or more. In other words, with a pressure drop of 20 MPa to obtain the (21) Poppe, H. J. Chromatogr., A 1997, 778, 3-21. (22) Bristow, P. A.; Knox, J. H. Chromatographia 1977, 10, 279-289.

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column efficiency of 15 000 theoretical plates with t0 of ∼10 s, one can expect that the performance of the monolithic silica column to be similar to that of a column packed with 2-µm particles. The monolithic column would provide higher performance than particle-packed columns if a greater number of theoretical plates was desired at this pressure drop. At a higher or lower limiting pressure, the critical number of theoretical plates that determines the choice between a particulate column and a monolithic column becomes greater or smaller, as shown later. The monolithic column MS(100)-T1.4-BII produced more than 20 000 theoretical plates in less than 20 s t0 at a pressure drop of ∼15 MPa, as shown in Figure 9, indicating that it is easily possible to produce 10 000 theoretical plates with less than 10 s of t0 with a pressure drop of less than 8 MPa by employing a shorter column. The column performance is close to the predicted performance of a column packed with 2-µm particles in this range of t0. Figure 11a shows a comparison between the monolithic silica columns and particle-packed columns at a 40 MPa pressure limit. The plots of log(t0/N2) against log(N) allow an easier comparison of the column performance in terms of column efficiency and separation time than Figures 8 or 10, as proposed by Desmet and co-workers.10 In Figure 11a, the curves for monolithic silica columns with smaller domains are shown below the curves representing the performance of particle-packed columns in a region where the number of theoretical plates is greater than ∼30 000, thus indicating superior performance for producing the number of theoretical plates (N) in this range. Although the optimum performance of these monolithic silica columns, which is much higher than that of a particle-packed column, is seen over a region of N greater than 100 000, they can compete with the performance of columns packed with 2-µm particles over the range over ∼30 000 at 40 MPa and over ∼15 000 at 20MPa (Figure 10). Figures 10 and 11a suggest that the older batch of monolithic silica MS(50)-D3 corresponds to a column packed with 3-4-µm particles, whereas the present monolithic silica columns are closer to those packed with 2-2.5-µm particles in performance at 15 000-30 000 theoretical plates under common pressures used in HPLC. The results indicate that the columns MS(100)-T1.4BI-MS(100)-T1.4-BIII showed performance in the “forbidden region” for the previous columns, except for the results obtained under ultra-high-pressure conditions.10 The comparison between the plots for the monolithic silica columns MS(100)-T1.4-BI-BIII and those for 1.4-, 2-, and 3-µm particles at 100 MPa (shown in Figure 11b) suggests that particlepacked columns perform better at N ) 80 000 theoretical plates or lower. At higher pressure drops, the comparison becomes more favorable for a particle-packed column. At 10 MPa, the monolithic silica column MS(100)-T1.4-BIII performs better than a particlepacked column at greater than 8000 theoretical plates, as shown in Figure 11c. Those factors which enabled the preparation of monolithic silica columns with higher homogeneity of the co-continuous structures are speculated to be the well-controlled phase separation and the gelation rates in the preparation,2 which enabled the formation of the uniform, small-sized domains. The numerous skeletons with increased phase ratios attached to the capillary wall seem to resist shrinkage. The preparation of monolithic silica becomes increasingly difficult with an increase in the capillary

Figure 11. Log(t0/N2) against log(N) for the columns evaluated. The curves for particle-packed columns were obtained by assuming the following parameters: η ) 0.00046 Pa s, φ ) 700, Dm ) 2.22 × 10-9 m2/s, and Knox equation, h ) 0.65ν1/3 + 2/ν + 0.08 ν. Maximum pressure: (a) 40, (b) 100, and (c) 10 MPa. The particle diameters for the particle-packed columns were 1.4, 2, 3, and 5 µm. The symbols are the same as in Figure 10 for the columns.

diameter. The preparation of a monolithic silica capillary column larger than 100-µm i.d. from TMOS has not been successful using the previous conditions.6 In the present study, the preparation of a 200-µm-i.d. column from TMOS was successful with increased phase ratios and small-sized domains. The MS(200)-T1.4-BII column showed slightly lower performance than the 100-µm-i.d. column, yielding H ) 6.3 µm at u ) 0.94 mm/s in 80% methanol. The development of large-sized capillary columns is important for the miniaturization of HPLC, because they can reduce the effects of extracolumn band broadening. A further study is in progress exploring the development of monolithic silica columns with even smaller domain sizes. It would be of great interest whether one

can prepare monolithic silica columns with a domain size of ∼1.52.0 µm or smaller with adequate structural homogeneity. CONCLUSIONS It was possible to prepare monolithic silica columns with increased phase ratios over that of the previous materials. These monolithic silica columns with increased phase ratios, small domains, and increased homogeneity showed performance higher than previous monolithic silica columns. They were comparable with columns packed with 2-2.5-µm particles for generating 15 000-30 000 or more theoretical plates under a pressure of 2040 MPa. Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Previous monolithic silica columns were able to show column efficiency of 3.5-4-µm particles at a pressure drop similar to a column packed with 7-10-µm particles. The present columns, which we would like to call second-generation monolithic silica columns, showed the performance of a column packed with 2-2.5µm particles with a pressure drop similar to those packed with 5-µm particles that can be conveniently used with common HPLC equipment. The predicted higher performance of monolithic silica columns with high homogeneity was realized.11 ACKNOWLEDGMENT This work was supported in part by Grants-in-Aid for Scientific Research funded by the Ministry of Education, Sports, Culture,

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Science and Technology, 14340234 and 17350036. The support from Nacalai-Tesque, GL Sciences, and Merck KGaA, Darmstadt are also gratefully acknowledged. NOTE ADDED AFTER ASAP PUBLICATION This article was released ASAP on October 12, 2006 with incorrect eta values in Figure captions 10 and 11. The correct version was posted on October 23, 2006.

Received for review April 26, 2006. Accepted August 28, 2006. AC060770E